Under appropriate circumstances the injection of an antigen into an animal produces a specific antiserum that reacts selectively with the antigen. This antiserum contains proteins that are responsible for the recognition of the antigen, i.e., proteins that possess a so-called "antibody function." Such proteins are commonly referred to as "antibodies" or, in a broader sense, as "immunoglobulins" (Ig).
All individuals within a given species have in common various Ig categories called "isotypes." For example, in humans 10 isotypes have been identified and grouped into five classes, namely IgG, IgM, IgA, IgD and IgE, which classes are further subdivided into subclasses, e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3 and IgG.sub.4 for Immunoglobulin G. These classes are also sometimes referred to or designated as .gamma.G, .gamma.M, .gamma.A, .gamma.D and .gamma.E, respectively. The predominant serum immunoglobulins are of the class IgG or gamma globulins.
Gamma globulin (IgG) preparations intended for injection into humans originally were produced by the alcohol fractionation procedure developed by Dr. E. Cohn and coworkers of Harvard during the 1940's: This procedure is described in Cohn et al., J. Amer. Chem. Soc. 68:459 (1946). These preparations were prepared as 10-18 wt-% protein solutions, the most common being a 15 wt-% protein solution. The latter was favored since at that concentration, the product exhibited the same viscosity as blood. Another advantage for these high protein gamma globulin products was their inherent shelf stability as judged by the absence of visually observable precipitation upon storage at 2.degree.-8.degree. C. for time periods of 2-3 years. In contrast, preparations containing less than about 10 wt-% protein frequently exhibited visually observable precipitation in the final liquid product.
The foregoing, relatively high protein gamma globulin products initially were intended for intravenous (IV) administration. The latter is considered advantageous clinically because immediate high levels of circulatory protective antibodies are attainable, whereas approximately one-half of the antibody given intramuscularly (IM) is lost due to local proteolysis and incomplete absorption. Unfortunately, experience has shown the above product not to be safe for IV administration due to adverse anaphylatic reactions in recipients. See, for example, Bowman, Clin. Obst. Gynec. 25:341 (1982) and Schroeder et al., Amer. J. Med., Mar. 30, 1984, page 33. The formation of aggregated gamma globulin polymers during storage of products prepared by the standard cold alcohol procedure allows for these aggregates to combine with complement in the patient's blood and to produce an anticomplement reaction. Thus, all gamma globulin products produced by the standard cold alcohol procedure have been approved only for intramuscular use.
The formation of relatively large molecular weight aggregates of gamma globulin in aqueous solutions is particularly deleterious to its pharmaceutical utility. Such denaturation is believed to be influenced by sulfhydryl-disulfide interchange reaction.
The ability of gammaglobulin to bind complement is greatly increased as a result of denaturation, in particular by aggregation to high molecular weight species. The complement binding mechanism of these aggregates appears to be identical to that of antigen-antibody complexes. Marcus, D. M., (1960) J. Immunol. 84:273-284. In the case of IgG, it is known that the complement binding site requires two molecules close together. It is therefore possible that critical packing of the molecules is required, rather than any necessary conformational change. However, the size distribution of aggregates in denatured antibody solutions and its relation to anticomplement activity has not been examined.
In earlier studies, Gerber, J. Immunol. 92:885-888(1964) and Gerber, Arthritis Rheum. 17:85-91(1971), it is postulated that in patients suffering from rheumatoid arthritis there is an in vivo mechanism triggered by metallic copper that causes increased formation of aggregate of gamma globulin. This worker found a beneficial effect to the patient if the level of one amino acid, L-histidine, was increased. These studies were done at physiological pH values, i.e, a pH of about 7.4 however. Also, the effect of histidine protection on relatively low protein human gamma globulin products in the area of absence of aggregate and anticomplementary activity was not examined in the studies by Gerber and cannot be predicted from the reported data.
As gamma globulin therapy has become an accepted practice, the demand for large quantities of commercial product worldwide has required manufacturers to adopt hyperimmunization programs for donors of the starting blood from which the gamma globulin-containing product is derived. The resulting plasma or serum has a desired higher starting antibody titer. Hence, more product can be derived from a fixed starting volume of blood. While the desired final antibody titer can also be attained at a lower protein concentration than the previous 10-18 wt-% value, such gamma globulin-containing products are less stable during storage.
Thus, one must either add protein to the product to maintain the desired 10-18 wt-% concentration, or develop methods for stabilizing a solution of the highly purified gamma globulin product having a protein concentration of less than 10 wt-%. For the former approach, it is common to add either purified albumin or gamma globulin derived from a low titer plasma source. However, this approach is not economically attractive. Also, it exposes the patient to additional health risks since viral contaminants from the additional purified proteins are a possibility. An example of the latter approach can be found in U.S. Pat. No. 4,186,196 to Lundblad et al. where a relatively high concentration of maltose is added to the final buffer to stabilize a 5 wt-% gamma globulin product. The latter is derived from a modified cold alcohol procedure that is said to remove aggregates as well, the objective being a product that can be administered intravenously.
Other prior attempts at stabilization of protein solutions are illustrated by U.S. Pat. No. 2,826,533 to Fowell which discloses the use of dextrose to increase the solubility of fibrinogen in solution, U.S. Pat. No. 4,089,949 to Thomas which discloses the use of a variety of carbohydrates (e.g., dextrose, mannose, galactose, fructose, lactose, sucrose and maltose) to enhance the solubility of an anti-hemophilic factor (AHF)-fibrinogin composition, and U.S. Pat. No. 3,057,781 to Mace et al. which discloses stabilization of plasma with invert sugar.
Other workers have explored the possibility of producing a gamma globulin product by an entirely different process, again with an objective to produce a product suitable for IM or IV administration. The approach currently favored employs ion-exchange chromatography. See, for example, U.S. Pat. No. 4,136,094 by Condie and Hoppe et al., Vox Sang. 25:308 (1973); Friesen et al., J. Applied Biochem. 3:164 (1981); and Walsh and O'Riordan, Irish Med. J. 75:232 (1982).
Condie contends that an IV administratable and stable 5 wt-% protein solution is possible without extra additives beyond the standard glycine-saline buffer. It should be noted, however, that a key step in this particular process is the pretreatment of the plasma source with colloidal silica. The present process, on the other hand, does not require exposure by workers to the potential health hazard of working with silica fumes while preparing a stabilized protein solution. The other three references of the foregoing grouping rely exclusively on the ion-exchange chromatography process to produce a final product said to be safe for IV administration. However, all three require that the final product, which contains very low protein concentration (1-4 wt-%), be lyophilized to meet adequate shelf stability requirements. The present invention, on the other hand, provides a relatively low cost procedure for amply stabilizing low protein gamma globulin solutions that avoids the inconvenience and expense of lyophilization and reconstitution prior to use.
To evaluate the stability of liquid gamma globulin products, a relatively simple test has been used historically. This test is recommended by the U.S. Bureau of Biologics and involves heating the finished product to a temperature of 57.degree. C. and holding it at that temperature for four hours while examining the product for visual precipitates. See Code of Federal Regulations 21, Food and Drugs, 640.101a (revised April 1978). Fernandes and Lundblad, Vox Sang. 39:101-112 (1980) report a modification of this procedure suitable for routine evaluation of potential additives. The modified procedure comprises heating approximately 2 milliliters of the test product at 57.degree. C. for four hours and then evaluating the percent change in degree of opalescence as measured by recording the transmittance at 580 nm with a laboratory spectrophotometer.
The preferred buffer for purified liquid gamma globulin products heretobefore has been glycine-saline, pH 6.4-7.2; however, this buffer has now been found to be inadequate if the protein concentration is 5 wt-% or less.
Another problem encountered with relatively low protein gamma globulin products which employ only glycine-saline as the stabilizer is pH control. Historically, the higher protein concentration (i.e. about 15 wt-%) has served as the principal buffering agent for the product, not either glycine or saline. Yet the relatively lower protein concentrations do not exhibit an adequate buffering effect.
The present invention, on the other hand, provides superior pH stabilization over the historical glycine-saline buffer for purified gamma globulin solution with a protein concentration of 5 wt-% or less.
Additionally, this invention provides a relatively low cost product which is safe when injected into patients for purposes of gamma globulin therapy. The present product is effective at much lower concentrations than the previously used, stabilized products, does not require the use of hazardous chemicals like silicon dioxide to treat the gamma globulin, and does not require chemical modifications of the gamma globulin itself.